Silicon, the second most abundant element on Earth, is widely used in the manufacture of siloxane-based semiconductors, glasses, ceramics, plastics, elastomers, resins, mesoporous molecular sieves and catalysts, optical fibers and coatings, insulators, moisture shields, photoluminescent polymers, and cosmetics [Auner, N. and Weis., J. (1998) Organosilicon Chemistry III: From Molecules to Materials, Wiley W C H; Auner, N. and Weis, J. Organosilicon Chemistry IV: From Molecules to Materials, Wiley W C H (in press); and Ball P. (1997) Made to Measure: New Materials for the 21st Century, Princeton University Press, Princeton, N.J., USA]. The Manufacture of these materials typically requires high temperatures or the use of caustic chemicals.
By contrast, the biological production of amorphous silica, the simplest siloxane [(SiO2)n], is accomplished under mild physiological conditions, producing a remarkable diversity of exquisitely structured shells, spines, fibers, and granules in many protists, diatoms, sponges, molluscs and higher plants [Simpson, T. L. and Volcani, B. E. (1981) Silicon and Siliceous Structures in Biological Systems, Springer-Verlag; and Voronkov, M. G., Zelchan, G. I. and Lukevits, E. J. (1997) Silicon and Life (2nd edn), Zinatne Publishing, Vilnius, Lithuania]. These biologically produced silicas exhibit a genetically controlled precision of nanoscale architecture that, in many cases, exceeds the capabilities of present-day human engineering. Furthermore, the biological production of siloxanes occurs on an enormous scale globally, yielding gigatons per year of silica deposits on the floor of the ocean. Diatomaceous earth (composed of the nanoporous skeletons of diatoms) is mined in great quantities from the vast primordial deposits of this biogenic silica.
Biotechnical approaches are now starting to unlock the molecular mechanisms of polysiloxane synthesis under physiological conditions, offering the prospect of new, environmentally benign routes to the synthesis and structural control of these important materials. Taking advantage of marine organisms that produce large relative masses of biogenic silica, molecular biologists have begun to isolate the genes and proteins controlling silica biosynthesis and nanofabrication.
Hildebrand and colleagues made a significant breakthrough by cloning and characterizing the cDNA encoding the first silicic-acid [Si(OH)4] transporter to be unequivocally identified [Hildebrand, M., Volcani, B. E., Gassman, W., and Schroeder, J. I. (1997) Nature 385, 688-689]. They showed, by analysis of the encoded protein and by injection of the mRNA (synthesized in vitro from the cloned cDNA) into Xenophus eggs, that the transporter protein forms a sodium-dependent transmembrane ion channel that mediates the transport of silicic acid. The action of this protein can account for the uptake of the silica precursor from the dilute pool of silicic acid in oceanic and fresh water, and similar transporters may pump silicic acid (or its conjugates) into the lumen of the silica-deposition vesicle (silicalamella), in which polycondensation (polymerization) is known to occur.
Krxc3x6ger and colleagues have cloned and characterized cDNAs encoding two families of protein (at least one of which is glycosylated) that contribute to the organic sheath surrounding the silica walls of a diatom [Krxc3x6ger, N., Bergsdorf, C. and Sumper, M. (1994) EMBO J. 13, 4676-4683; and Krxc3x6ger, N., Lehmann, G., Rachel, R. and Sumper, M. (1997) Eur. J. Biochem. 250, 99-105.]. The proteins most intimately associated with these silica walls contain regularly repeating hydroxyl-rich domains potentially capable of interacting with the growing silica structure [Hecky, R. E., Mopper, K., Kilham, P., and Degens, E. T. (1973) Mar. Biol. 19,323-331; Swift, D. M. and Wheeler, A. P. (1992) Phycology 28, 209-213; and Harrison, C. C. (1996) Phytochemistry 41, 37-42]. Hecky et al. had proposed that such hydroxyl-rich domains might align silicic-acid monomers, either by condensing with them (with elimination of water) to form covalent adducts or by hydrogen bonding, thus bringing them into favorable juxtaposition for their condensation to form silica. Thermodynamic calculations support the energetic feasibility of such a pathway [Lobel, K. D., West, J. K., and Hench, L. L. (1996) Mar. Biol. 126, 353-360].
Other researchers have suggested that various organic conjugates of silicic acid might serve as the proximate substrates for polymerization in vivo. Silicon catecholates have been used by Perry et al. in extensive studies of silica polymerization promoted by sugars and polysaccharides from silicified plants (Harrison, C. C. (1996) Phytochemistry 41, 37-42; Harrison, C. C., and Loton, N. J. (1995) J. Chem. Soc.-Faraday Trans. 91, 4287-4297; and Perry, C. C. and Yun, L. J. (1992) J. Chem. Soc.-Faraday Trans. 88, 2915-2921), and Mann and his colleagues recently showed that bacterial filaments can direct the deposition of a colloidal silica gel which after calcination yielded a macroporous filamentous material [Davis, S. A., Burkett, S. L., Mendelson, N. H., and Mann, S. (1997) Nature 385, 420-423].
In contrast to anthropogenic and geological syntheses of these materials that require extremes of temperature, pressure or pH, living systems produce a remarkable diversity of nanostructured silicates at ambient temperatures and pressures and at near-neutral pH. Laboratory methods have been unable to replicate these results and rely instead on extreme pHs and/or surfactants to condense silica precursors into specific morphologies or patterned structures. These conditions are undesirable for environmental reasons and therefore methods to direct silica assembly under conditions similar to those used in nature (i.e. biomimetically) are desired.
The present invention overcomes the drawbacks of prior efforts to condense silica precursors into specific morphologies or patterned structures, and provides heretofore unattainable materials having very desirable and widely useful properties. These materials are prepared at ambient temperatures and pressures and at near-neutral pH.
The method of the present invention for in vitro polymerization of silica and silicone polymer networks, includes the steps of (1) combining a catalyst and a substrate, wherein the substrate is selected from the group consisting of silicon alkoxide, metal alkoxide, and organic conjugates of the foregoing; and (2) polymerizing the substrate to form silica, polysiloxanes, polymetallo-oxanes, or mixed poly(silicon/metallo)oxane materials at about neutral pH. Preferably the substrate is a silicon alkoxide having the general formula Rxe2x80x94Sixe2x80x94(Oxe2x80x94Et)3, wherein Et is ethyl and R is methyl, phenyl, or ethoxy. Moreover, the polymerized materials preferably include apolysilsesquioxame having the general formula (RSiO3/2)n, wherein n is an integer greater than 1.
The present invention also provides compositions for use in polymerizing silica and silicone polymer networks, which includes a silicon alkoxide substrate; and a catalyst that assembles, hydrolyzes, and condenses the substrate at about neutral pH. A catalyst according to the present invention is generally a protein or polypeptide. Preferred protein or polypeptide catalysts include silicatein filaments, silicatein subunits, cysteine homopolymers, and cysteine-containing block copolypeptides. A preferred silicatein is a protein comprising an amino acid sequence at least 70% identical to the amino acid sequence of silicatein xcex1, i.e., SEQ ID NO:1. Alternatively the catalyst is a recombinant protein encoded by a nucleotide sequence at least 70% identical to the coding regions of SEQ ID NO:2, which is the cDNA sequence of the silicatein xcex1 gene. Yet another group of catalysts of the present invention, which unexpectedly mimic the polymerizing and scaffolding activities of silicateins, are cysteine-containing block copolypeptides. The most preferred versions the diblock copolypeptides are poly(L-Cysteine10-b-L-Lysine200), poly(L-Cysteine30-b-L-Lysine200), poly(L-Cysteine60-b-L-Lysine200), and poly(L-Cysteine30-b-L-Lysine400).
Silicified structures can be synthesized according to the method of the present invention. These structures assume a shape directed by the scaffolding activity of the catalyst. Such silicified structures can include shapes, such as filaments, spheres, elongated globules, and columns.